This competing renewal application builds on findings from the initial funding period that establish COX-2 as a critical signal for fracture repair. The role of COX-2 extends to fracture healing in aging. Decreased COX-2 expression is observed in the fracture callus of aged mice and preliminary data show that the reduced fracture repair observed in these animals can be rescued by delivery of an agonist targeting the PGE2 receptor, EP4. Based on our finding that COX-2 is induced specifically in mesenchymal chondroprogenitors and immature chondrocytes during early endochondral bone healing, the proposal addresses the role of mesenchyme specific and chondrocyte specific COX-2/EP4 receptor expressions during fracture healing using conditional gene deletion technology. In addition, we will also define the signaling mechanisms downstream of COX-2 by which Indian Hedgehog (Ihh) is induced by COX-2/PGE2 via activation of ATF4 phosphorylation. Our overall hypotheses are 1) induction of COX-2 in early fractures by mesenchymal stem cells and immature chondrocytes plays a critical role in bone repair; 2) COX-2/EP4 signaling targets cartilage and activates the Ihh pathway, which is essential for chondrocyte proliferation, early vascular invasion, and the transition from cartilage to bone formation. In Aim1, we will characterizes fracture healing in COX-2f/f; Prx-1Cre and EP4f/-;Prx1Cre mice, in which COX-2 or EP4 receptor are deleted in mesenchymal cells or chondrocytes respectively. In Aim2, we will examine the signaling mechanisms by which Ihh is induced via activation of ATF4 phosphorylation downstream COX-2/PGE2. In Aim3, important translational studies will definitively establish that EP4 agonists can compensate for the reduced fracture repair observed in aging. Related in vivo approaches with HH gain and loss of function will further establish a role for hedgehog signaling downstream of COX2/PGE2/EP4 during fracture healing. This studies will advance our current limited knowledge regarding mesenchymal cell initiated endochondral bone repair, with the potential to provide novel therapeutic approaches to treat elderly patients with impaired fracture healing.